Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System

Undramaa Bat-Erdene, John M. Billingsley, William C. Turner, Benjamin R. Lichman, Francesca M. Ippoliti, Neil K. Garg, Sarah E. O'Connor, Yi Tang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD+-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD+ to NADH and a low ratio of NADP+ to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450, and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD+-dependent dehydrogenase to the one-pot mixture led to ∼1 g/L of nepetalactone, the active cat attractant in catnip.

Original languageEnglish
Pages (from-to)9898-9903
Number of pages6
JournalACS Catalysis
Issue number15
Early online date23 Jul 2021
Publication statusPublished - 6 Aug 2021

Bibliographical note

Funding Information:
This work is support by NIH R01AT010001 to Y.T. The plasmid used for expression of NoxE was a gift from Prof. James Bowie.

© 2021 American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details


  • biocatalysis
  • cofactor regeneration
  • enzyme cascade
  • natural products
  • synthetic biochemistry

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